Western blot analyses were carried out as previously described (13) . Briefly, cells were lysed with 1% Triton X-100 or NP-40 with 1X protease inhibitor cocktail (MCE, HY-K0010). Lysates were resolved on SDS-PAGE followed by immunoblotting.
Anti-Histone H3 (Cell Signaling Technology, cat. #4499),
Anti-Histone H3K27me3 (Cell Signaling Technology, cat. #9733), and other primary antibodies were used. When indicated, cell were treated with an EZH2 inhibitor GSK-126 (MCE, HY-13470) or agonist GSK-J4 (MCE, HY-15648B) for 7 to 14 days. For immunoprecipitation experiments, whole-cell lysates were precleared with protein A/G beads (MCE, HY-K0202) and incubated with IgG control or anti-AcK antibody (1:100, Cell Signaling Technology, 9441) for overnight rocking at 4 C. Protein G beads were added and mixed for another 4 hours. For anti-avi immunoprecipitation experiments, lysates from cells coexpressing Escherichia coli biotin holoenzyme synthetase (BirA) and indicated proteins were incubated with
streptavidin-conjugated magnetic beads (Promega, Z5482) for 4-hour rocking at 4 C. Immunocomplexes were resolved on SDS-PAGE, followed by immunoblotting.
Liao K., Deng S., Xu L., Pan W., Yang S., Zheng F., Wu X., Hu H., Liu Z., Luo J., Zhang R., Kuang D.M., Dong J., Wu Y., Zhang H., Zhou P., Bei J.X., Xu Y., Ji Y., Wang P., Ju H.Q., Xu R.H, & Li B. (2020). A Feedback Circuitry between Polycomb Signaling and Fructose-1, 6-Bisphosphatase Enables Hepatic and Renal Tumorigenesis. Cancer research, 80(4).
